Development of lead ion testing paper with naked-eye observable readout for ten min on-site detection

ABSTRACT

A luminescent Ir(III) complex is used to develop a label-free G-quadruplex-based assay for lead ions in liquid or solution. In particular, the present invention describes method for monitoring lead ion concentration in water.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Continuation-in-Part Application of U.S. non-provisionalpatent application Ser. No. 15/291,041 filed on Oct. 11, 2016 whichclaims benefit from U.S. provisional application No. 62/240,502 filedOct. 12, 2015, and the disclosure of which is incorporated herein byreference.

FIELD OF INVENTION

The present invention relates to the synthesis and use of a luminescentiridium(III) complex for the construction of a label-freeG-quadruplex-based assay for the rapid detection of lead ions in asolution. In particular, the present invention relates to method formonitoring lead ion concentration in water using a label-freeG-quadruplex-based assay implemented on a paper-based chip.

BACKGROUND OF INVENTION

Heavy-metal pollution has attracted much attention in the public mediaand scientific community due to the toxic effects of heavy metal ions onhuman health and the environment. The lead(II) ion, as one of theacutely toxic metal ions, is a dangerous contaminant which causesadverse health effects in humans, including delayed physical and mentaldevelopmental in infants and children, kidney disease and high bloodpressure in adults. Meanwhile, certain oligonucleotides undergoconformational changes in the presence of particular heavy metal ions.

Atomic absorption spectrometry (AAS) and inductively coupled plasma massspectrometry (ICP-MS) are widely-used instrumental techniques forlead(II) ion detection, but their sophisticated instrumentation and/orcomplicated sample preparation hamper their application for in-fieldstudies. In recent years, fluorescent, colorimetric methods utilizingorganic fluorescent dyes, oligonucleotides, or lead(II)-dependent RNAcleaving DNA enzymes have been developed for lead(II) ion detection. Ashort testing time and portable instrument or tool is required for rapidonline detection of lead ions in drinking water around the standard ofWorld Health Organization (WHO) for safe drinking water.

SUMMARY OF INVENTION

In accordance with a first aspect of the present invention, aluminescent iridium(III) (Ir(III)) complex is utilized to construct alabel-free G-quadruplex-based assay for the rapid detection of lead ionsin a solution or liquid. In particular, the present invention providesan application of the assay for monitoring lead ion concentration inwater.

In one embodiment, the present invention provides a system for detectinglead ions in a liquid comprising

-   -   at least one luminescent iridium(III) complex and    -   at least one G-quadruplex-forming sequence;        -   wherein said G-quadruplex-forming sequence is a single            stranded oliogmer in the absence of lead ions and said            G-quadruplex-forming sequence forms a G-quadruplex structure            in the presence of lead ions and wherein said            G-quadruplex-forming sequence reacts with said at least one            luminescent iridium(III) complex to emit luminescent            emission. In one embodiment, the at least one luminescent            iridium(III) complex is Ir(III) complex,            [Ir(epyd)₂(dclphen)]PF₆ wherein            epyd=2-(4-ethylphenyl)pyridine;            dclphen=4,7-dichloro-1,10-phenanthroline and said at least            one G-quadruplex-forming sequence is PS2.M,            5′-GTGGGTAGGGCGGGTTGG-3′ (SEQ ID NO. 1).

In one embodiment, said liquid is water.

The luminescent emission is measured using at least one spectrometer.

In another embodiment, the system further comprises a detection chip,

Said detection chip comprises a plurality of superhydrophobic areas anda plurality of hydrophilic areas, wherein said hydrophilic areascomprise one or more detection zones where said G-quadruplex-formingsequence is stored, one of more storage zones where said luminescentiridium(III) complex is stored and one or more connected zones thatconnect the detection and storage zones,

-   -   wherein said superhydrophobic areas comprise one or more        hydrophobic valves located within said connected zones which        separate the detection and storage zones;    -   and wherein said one or more hydrophobic valves is removable to        allow flow of said at least one luminescent iridium(III) complex        from the at least one storage zones to the at least one        detection zones.

In a second aspect, the present invention provides a method fordetecting lead ions in a liquid comprising providing the system of thepresent invention,

-   -   introducing said liquid to the G-quadruplex-forming sequence to        form a first mixture;    -   adding the luminescent iridium(III) complex to the first mixture        to form a second mixture; and    -   Measuring luminescent emission of the second mixture.

In one embodiment, the method further comprising incubating the firstmixture at temperature of 32° C. to 99° C. and cooling the first mixtureat −18° C. to 32° C.

In another embodiment, the method includes introducing said liquid tothe detection zone of the detection chip; removing the hydrophobicvalves; and measuring luminescent emission of the detection chip.

In another embodiment, the detection chip is a paper substrate. Saidsuperhydrophobic areas are coated by imprinting a solution of a PDMSprepolymer and a curing agent mixed in 10:1 w/w ratio and the one ormore hydrophobic valves comprise polytetralfluoroethylene.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described.

The invention includes all such variation and modifications. Theinvention also includes all of the steps and features referred to orindicated in the specification, individually or collectively, and anyand all combinations or any two or more of the steps or features.

Other aspects and advantages of the invention will be apparent to thoseskilled in the art from a review of the ensuing description.

BRIEF DESCRIPTION OF DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings, in which:

FIG. 1 shows chemical structure of the luminescent Ir(III) complex,complex 1, of the present invention.

FIG. 2 shows luminescence response of complex 1 with different DNAsequences.

FIG. 3 shows luminescence spectra of complex 1/G4-quadruplex system inthe presence of lead ions (10 μg/L) or absence of lead ions (0 μg/L)using NanoDrop 3300 spectrometer by deducting the complex1/G4-quadruplex system background luminescence.

FIG. 4 shows luminescence spectra of complex 1/G4-quadruplex system inresponse to various concentrations of lead ions: 10, 20, 30, 100 and1000 μg/L spiked into MiliQ™ ultrapure water using NanoDrop 3300spectrometer by deducting the complex 1/G4-quadruplex system backgroundluminescence.

FIG. 5 shows luminescence spectra of complex 1/G4-quadruplex system inresponse to various concentrations of lead ions: 10, 20, 30, 100 and1000 μg/L spiked into tap water using NanoDrop 3300 spectrometer bydeducting the complex 1/G4-quadruplex system background luminescence.

FIG. 6 shows the diagrammatic bar array representation of theluminescence enhancement selectivity ratio of complex 1 [I/I₀] for PS2.M G-quadruplex DNA over double strand DNA (dsDNA, ds26) and singlestrand DNA (ssDNA, ds17.2). [PS2. M G-quadruplex DNA over double strandDNA (dsDNA, ds26)=IPS2.M/IdsDNA and PS2. M G-quadruplex DNA over singlestrand DNA (ssDNA, ds17.2)=IPS2.M/IssDNA]

FIG. 7 shows the schematic diagram of the luminescent switch-on assay tomonitor lead ions using the G-quadruplex-selective probe complex 1.

FIG. 8 shows the schematic diagram of paper-based detection of leadions.

FIG. 9 shows the schematics of the paper-based chip. (A) shows thefabrication process of the paper-based chip. (B) shows the schematics ofthe paper-based chip, which consists of two zones (detection zone andstorage zone) and a hydrophobic Teflon sticker.

FIG. 10 shows a paper-based chip fabricated using the protocol shown inFIG. 9.

FIG. 11 shows a schematic diagram of the luminescent switch-on assaythat monitors the presence of lead ions using the G-quadruplex-selectiveprobe complex 1 on a paper testing strip (The dot of the right stripwith solid particles in the middle of the detection zones represents thepresence of lead ions in the water sample exceeding a pre-specifiedlimit).

FIG. 12 shows the fluorescence images of different concentrations ofPb²⁺ ions (nM).

FIG. 13 shows the fluorescence intensity of different concentrations ofPb²⁺ ions (nM).

FIG. 14 shows a portable device for the point of care testing of Pb²⁺,the results can be captured by a smart phone.

DETAILED DESCRIPTION OF INVENTION

The present invention is not to be limited in scope by any of thespecific embodiments described herein. The following embodiments arepresented for exemplification only.

Without wishing to be bound by theory, the present invention provides aluminescent Ir(III) complex for constructing a label-freeG-quadruplex-based assay for the rapid detection of lead ions in aliquid or solution. In particular, the present invention provides amethod for monitoring lead ion concentration in water.

The lead(II) ion is known to induce the structural transition of aguanine-rich single-stranded oligonucleotide into a G-quadruplex, whichis a non-canonical DNA secondary structure consisting of planar stacksof four guanines stabilized by Hoogsteen hydrogen bonding. Meanwhile,rapid advances in the field of DNA technology over the past severalyears have highlighted the potential use of oligonucleotides asattractive signal transducing units for the detection of biological andenvironmental important analytes. Oligonucleotides offer salientadvantages in bio-sensing applications, such as their relatively smallsize, low cost, facile synthesis and modification, good thermalstability, and reusability. In particular, the G-quadruplex motif, whichis a non-canonical DNA secondary structure composed of planar stacks offour guanines stabilized by Hoogsteen hydrogen bonding, has attractedparticular interest in sensing applications. The extensive structuralpolymorphism of G-quadruplexes has rendered them as versatilesignal-transducing elements for the development of DNA-based probes. Inrecent years, a number of oligonucleotide-based sensing platforms forlead ions have been developed. For example, Zhou and co-workers, ChinaPatent CN103305622A; and Sun and co-workers, China Patent CN103792229A,have reported fluorescent assays for lead ions by utilizing G-richoligomer and fluorescent organic dyes. The invention from Zhou andco-workers uses SYBR Green I, which is a commercially-available organicdye with low selectivity for G-quadruplex DNA, and also has narrow Stokeshifts and short lifetimes when compared to transition metal complexes.Also, SYBR Green I may be influenced by other compounds in the realwater sample or organic tissues. Moreover, the assay is a switch-offmethod which might lead to false positive results, and also requires atleast 20 minutes for measuring each sample. The other invention from Sunand co-workers employs a reaction-based system to detect lead ions, inwhich lead ions act as an inhibitor of a reaction. The detection limitof this invention in real water sample is unknown, and the tested valuein urine could not reach the standard of World Health Organization (WHO)for safe drinking water (below 10 μg/L). Guo, Fu and co-workers,Biosens. Bioelectron., 2012, 35, 123-127 have utilized a fluorescentorganic dye N-methyl mesoporphyrin IX (NMM) to detect lead ions byG-quadruplex based label-free platform. In the presence of lead ions,the DNA conformation does not bind the fluorescent organic dye, leadingto a switch-off response when the concentration of lead ions isincreased. This method also needed 30 minutes to measure a sample.Additionally, Tao Li, Erkang Wang and Shaojun Dong, Anal Chem., 2010,82, 1515-1520 and J. Am. Chem. Soc., 2010, 132, 13156-13157 have alsoutilized DNA conformational change to monitor lead ions. These reportsdemonstrate that DNA oligonucleotides can be integrated as useful,functional and structural elements for the construction of sensitiveluminescent platforms for the detection of lead ions. However, many ofthese methods still are unable to detect the presence of low levels oflead ions in aqueous samples. Herein, the present invention provides asystem using luminescent transition metal complex in conjunction with aG-quadruplex-based platform (Biosens. Bioelectron., 2013, 41, 871-874).This platform can be used directly on liquid or aqueous samples and thetesting period is dramatically decreased to less than 10 minutes usingthe specified method and a portable instrument, which is a uniquecombination that is used in the present invention.

In recent years, luminescent transition metal complexes have arisen asviable alternatives to organic dyes for sensory applications due totheir notable advantages. Firstly, metal complexes generally emit in thevisible region with a long phosphorescence lifetime, allowing them to bereadily distinguished from a fluorescent background arising fromendogenous fluorophores in the sample matrix by the use of time-resolvedfluorescent spectroscopy. Secondly, the precise and versatilearrangement of co-ligands on the metal centre allows the interactions ofmetal complexes with biomolecules to be fine-tuned for maximumselectivity and sensitivity. Thirdly, these metal complexes oftenpossess interesting photophysical properties that are strongly affectedby subtle changes in their local environment. For example, platinum(II)(Pt(II)) and ruthenium(II) (Ru(II)) complexes have been extensivelyinvestigated as “molecular light switches” for nucleic acids, includingG-quadruplex DNA. However, luminescent complexes based on the Ir(III)center have been comparatively less explored. In this invention, aluminescent Ir(III) complex is synthesized and evaluated for its abilityto act as G-quadruplex-selective luminescence switch-on probe. TheIr(III) complex 1, [Ir(epyd)₂(dclphen)]PF₆ (whereepyd=2-(4-ethylphenyl)pyridine;dclphen=4,7-dichloro-1,10-phenanthroline) (FIG. 1) is employed as aG-quadruplex probe for the construction of a label-free luminescentrapid detection platform for lead ions in liquid or solution. Also, theapplication of this platform for monitoring lead ions in water in ashort time period is demonstrated. To our knowledge, no luminescentG-quadruplex-based assay for the rapid detection of lead ions in thewater has yet been reported. And the metal complex of the presentinvention is designed with the superiority in binding G-quadruplex basedon our experience of developing G-quadruplex-selectivity probe (Acc.Chem. Res., 2014, 47, 3614-3631).

Results and Discussion

Principle of Luminescent G-Quadruplex-Based Probe for Lead Ion Detection

Lead ions can induce a guanine-rich single-stranded DNA (ssDNA)oligonucleotide into a G-quadruplex motif (Org. Lett., 2000, 2,3277-3280 and J. Mol Biol, 2000, 296, 1-5). The mechanism of lead ionrapid detection platform is outlined in FIG. 7. One embodiment of thepresent invention uses a single strand oligomer consisting of aG-quadruplex-forming sequence (PS2.M, 5′-GTGGGTAGGGCGGGTTGG-3′), whichacts as a special sensor for lead ions. In the absence of lead ions, thesingle-strand oligonucleotide, PS2.M, will not fold into G-quadruplexstructure, and remains as a single-strand structure that interacts onlyweakly with the luminescent Ir(III) complex. In contrast, in thepresence of lead ions, PS2.M folds into a G-quadruplex structure. Thenascent G-quadruplex structure is then recognized by the luminescentIr(III) complex with an enhanced emission response, allowing the systemto function as a switch-on luminescent probe for lead ions.

Verification of Ir(III) Complex 1 as G-Quadruplex-Selective Probe

In the present invention, emission response of luminescent Ir(III)complex 1 (FIG. 1) to different forms of DNA, including G-quadruplex,ssDNA and dsDNA (Table S1), is shown. Complex 1 bearing the N^N liganddclphen (4,7-dichloro-1,10-phenanthroline) and the C^N ligand epyd(2-(4-ethylphenyl)pyridine) shows a selective response for G-quadruplexDNA (FIG. 2 and FIG. 6), while not showing any luminescence enhancementtowards lead ions (data not shown). This result demonstrates the abilityof complex 1 to discriminate between G-quadruplex DNA, dsDNA and ssDNA.The luminescence enhancement of complex 1 in the presence ofG-quadruplex DNA is believed to be due to its ability to bind toG-quadruplex structures through groove/loop binding or end-stackinginteractions. This shields the complex from the aqueous solventenvironment and suppresses non-radiative decay of the excited state,thus leading to enhanced triplet state emission.

Luminescent Rapid Detection of Lead Ions in Liquid

The characterization and photophysical properties of the Ir(III) complex1 are given in the Table S2. Given the promising G-quadruplex-selectiveluminescent behaviour exhibited by complex 1, one embodiment of thepresent invention sought to employ complex 1 as a G-quadruplex-selectiveprobe and PS2.M as the lead ion sensor to construct a label-freeluminescent rapid detection platform for lead ions in aqueous solution.The luminescence response of complex 1 and PS2.M to lead ions is shown.Upon incubation with lead ions, the luminescence of complex 1 issignificantly enhanced. The luminescence enhancement of is due to theformation of the G-quadruplex structure of PS2.M induced by lead ions,which is subsequently recognized by complex 1 (FIG. 3 and FIG. 4). Theseresults suggest that the luminescence enhancement of the systemoriginated from the specific interaction of complex 1 with theG-quadruplex motif, PS2.M, which is generated by the structuraltransition of PS2.M, in the presence of lead ions.

Application of Lead Ion Detection Assay in Water

The performance of the G-quadruplex-based sensing platform of thepresent invention to detect lead ions in tap water is shown. In areaction system containing tap water, the present complex 1/G-quadruplexDNA system experiences a gradual increase in luminescence intensity withincrease concentration of lead ions (FIG. 5). This result demonstratesthat this sensing system of the present invention is useful for rapiddetection of lead ions in water.

Experimental Section

Materials

Reagents, unless specified, are purchased from Sigma Aldrich (St. Louis,Mo.) and used as received. Iridium chloride hydrate (IrCl₃.xH₂O) ispurchased from Precious Metals Online (Australia). All oligonucleotidesare synthesized by Techdragon Inc. (Hong Kong, China).

General Experimental

Mass spectrometry is performed at the Mass Spectroscopy Unit at theDepartment of Chemistry, Hong Kong Baptist University, Hong Kong(China). Deuterated solvents for Nuclear magnetic resonance (NMR)purposes are obtained from Armar and used as received.

¹H and ¹³C NMR are recorded on a Bruker Avance 400 spectrometeroperating at 400 MHz (¹H) and 100 MHz (¹³C). ¹H and ¹³C chemical shiftsare referenced internally to solvent shift (acetone-d₆: ¹H δ 2.05, ¹³C δ29.8; CD₃Cl: ¹H δ 7.26, ¹³C δ 76.8). Chemical shifts (δ are quoted inppm, the downfield direction being defined as positive. Uncertainties inchemical shifts are typically ±0.01 ppm for ¹H and ±0.05 for ¹³C.Coupling constants are typically ±0.1 Hz for ¹H-¹H and ±0.5 Hz for¹H-¹³C couplings. The following abbreviations are used for conveniencein reporting the multiplicity of NMR resonances: s, singlet; d, doublet;t, triplet; q, quartet; m, multiplet; br, broad. All NMR data isacquired and processed using standard Bruker software (Topspin).

Photophysical Measurement

Emission spectra and lifetime measurements for complexes are performedon a PTI TimeMaster C720 Spectrometer (Nitrogen laser: pulse output 337nm) fitted with a 380 nm filter. Error limits are estimated: λ (±1 nm);τ (±10%); φ (±10%). All solvents used for the lifetime measurements aredegassed using three cycles of freeze-vac-thaw.

Luminescence quantum yields are determined using the method of Demas andCrosby [Ru(bpy)₃][PF₆]₂ in degassed acetonitrile as a standard referencesolution (Φ_(r)=0.062) and calculated according to the followingequation:Φ_(s)=Φ_(r)(B _(r) /B _(s))(n _(s) /n _(r))²(D _(s) /D _(r))

where the subscripts s and r refer to sample and reference standardsolution respectively, n is the refractive index of the solvents, D isthe integrated intensity, and Φ is the luminescence quantum yield. Thequantity B is calculated by B=1-10^(−AL), where A is the absorbance atthe excitation wavelength and L is the optical path length.

Synthesis

The following complex is prepared according to (modified) literaturemethods. All complexes are characterized by ¹H NMR, ¹³C NMR, highresolution mass spectrometry (HRMS) and elemental analysis.

The precursor Ir(III) complex dimer [Ir₂(C^N)₄Cl₂] is prepared(C^N=epyd=2-(4-ethylphenyl)pyridine). Then, a suspension of[Ir₂(C^N)₄Cl₂] (0.2 mmol) and corresponding N^N ligand dclphen(dclphen=4,7-dichloro-1,10-phenanthroline, 0.44 mmol) in a mixture ofDCM:methanol (1:1, 20 mL) is refluxed overnight under a nitrogenatmosphere. The resulting solution is then allowed to cool to roomtemperature, and filtered to remove unreacted cyclometallated dimer. Tothe filtrate, an aqueous solution of ammonium hexafluorophosphate(excess) is added and the filtrate is reduced in volume by rotaryevaporation until precipitation of the crude product occurred. Theprecipitate is then filtered and washed with several portions of water(2×50 mL) followed by diethyl ether (2×50 mL). The product isrecrystallized by acetonitrile: diethyl ether vapor diffusion to yieldthe titled compound.

Complex 1. Yield: 68.7%. ¹H NMR (400 MHz, Acetone-d₆) δ 8.72 (d, J=1.2Hz, 2H), 8.42 (d, J=5.6 Hz, 2H), 8.25 (d, J=5.6 Hz, 2H), 8.18 (d, J=8.0Hz, 2H), 7.91-7.83 (m, 2H), 7.79 (d, J=0.8 Hz, 2H), 7.76 (d, J=0.8 Hz,2H), 6.96-6.92 (m, 4H), 6.29 (d, J=1.2 Hz, 2H), 2.43 (d, J=7.6 Hz, 4H),1.03 (t, J=7.6 Hz, 6H); ¹³C NMR (100 MHz, Acetone-d₆) 168.7, 152.7,150.5, 148.9, 147.4, 145.7, 142.8, 139.4, 131.9, 131.0, 128.7, 126.3,125.8, 123.8, 123.4, 120.4, 29.8, 15.5; MALDI-TOF-HRMS: Calcd forC₃₈H₃₀Cl₂IrN₄[M-PF₆]⁺: 805.1477. Found: 805.5739; AnaL:(C₃₈H₃₀Cl₂IrN₄PF₆+2H₂O) C, H, N: calcd 46.25, 3.47, 5.68; found. 46.04,3.11, 5.67.

Luminescence Response of Ir(III) Complex 1 Towards Different Forms ofDNA

The G-quadruplex DNA-forming sequence (PS2. M), dsDNA or ssDNA isannealed in Tris-HCl buffer (10 mM Tris, 100 mM KCl, pH 7.1) and isstored at −20° C. before use. Complex 1 (1 μM) is added to 5 μM ofssDNA, dsDNA or PS2. M G-quadruplex DNA in Tris-HCl buffer (10 mM Tris,pH 7.1). Emission spectra are recorded in the 460-770 nm range using anexcitation wavelength range of between 200-400 nm using PTI TimeMasterC720 Spectrometer.

Luminescent Rapid Detection of Lead Ions in Liquid

1 μL of ultrapure water sample with different concentrations of spikedlead ions and 1 μL of the G-quadruplex DNA-forming sequence (PS2. M:5′-GTGGGTAGGGCGGGTTGG-3′, 100 μM) are added to 8 μL of Tris buffer (10mM Tris, pH=7.9) in a microtube;

1 μL of pure water sample and 1 μL of the G-quadruplex DNA-formingsequence (PS2. M, 100 μM) are added to 8 μL of Tris buffer (10 mM Tris,pH=7.9) in a microtube, which was used as a blank sample;

-   -   The mixtures of blank and tap water samples are annealed at        least 1 minute under temperature ranging between 32° C. to 99°        C., preferably 32.1° C. to 98.6° C.;    -   After heating, blank and tap water samples are then cooled down        for at least 30 seconds under temperature ranging between        −18° C. to 32° C. Meanwhile, the NanoDrop 3300 spectrophotometer        is initiated;    -   0.3 μL of designed probe, Ir(III) complex, (0.5 μM) is added        into blank sample. After mixing, 1-2 μL of the mixed solution is        put onto the sample pool of NanoDrop 3300 spectrometer for blank        correction;

0.3 μL of designed probe, Ir(III) complex, (0.5 μM) is added into tapwater sample. After mixing, 1-2 μL of mixed solution is put onto thesample pool of NanoDrop 3300 spectrometer for measurement. Emissionspectra are recorded in the 440-750 nm range using an excitationwavelength range of between 200-400 nm. The peak demonstrates theexceeding level of lead ions in spiked ultrapure water sample (FIG. 3and FIG. 4).

Application of Lead Ion Rapid Detection Assay in Monitoring DrinkingWater

-   -   1-1.5 mL of water sample is collected from tap water;    -   1 μL of tap water sample with different concentrations of lead        ions and 1 μL of the G-quadruplex DNA-forming sequence (PS2. M:        5′-GTGGGTAGGGCGGGTTGG-3′, 100 μM) are added to 8 μL of Tris        buffer (10 mM Tris, pH=7.9) in microtube;    -   1 μL of pure tap water sample and 1 μL of the G-quadruplex        DNA-forming sequence (PS2. M, 100 μM) are added to 8 μL of Tris        buffer (10 mM Tris, pH=7.9) in microtube, which is used as a        blank sample;    -   The mixtures of blank and tap water samples are annealed at        least 1 minute under temperature ranging between 32° C. to 99°        C., preferably 32.1° C. to 98.6° C.;    -   After heating, blank and tap water samples are then cooled down        for at least 30 seconds under temperature ranging between        −18° C. to 32° C. Meanwhile, the NanoDrop 3300 spectrophotometer        is initiated;    -   0.3 μL of designed probe, Ir(III) complex 1, (0.5 μM) is added        into blank sample. After mixing, 1-2 μL of mixed solution is put        onto the sample pool of NanoDrop 3300 spectrometer for blank        correction;

0.3 μL of designed probe, Ir(III) complex 1, (0.5 μM) is added into tapwater sample. After mixing, 1-2 μL of mixed solution is put onto thesample pool of NanoDrop spectrometer for measurement. Emission spectrawere recorded in the 440-750 nm range using an excitation wavelengthrange of between 200-400 nm. The peak demonstrates the exceeding levelof lead ions in tap water (FIG. 5).

TABLE S1 DNA sequences used in the present invention: Sequence PS2.M5′-GTGGGTAGGGCGGGTTGG-3′ (SEQ ID No. 1) ds265′-CAATCGGATCGAATTCGATCCGATTG-3′ (SEQ ID No. 2) ds17.25′-GGGTTACTACGAACTGG-3′ (SEQ ID No. 3)

TABLE S2 Photophysical properties of Ir(III) complex 1. UV/visabsorption Com- Quantum λ_(abs)/nm (ε/ plex yield λ_(em)/nm Lifetime/μsdm³ · mol⁻¹ · cm⁻¹) 1 0.063 639 4.577 ± 5.090 × 10⁻³ 267 (7.95 × 10³)

Summaries

The ability of luminescent Ir(III) complex containing epyd C^N anddclphen N^N ligands to act as a G-quadruplex probe is shown. The Ir(III)complex 1 is discovered to be a G-quadruplex-selective luminescentprobe, and a label-free luminescent assay for lead ions is provided inthe present invention utilizing the G-quadruplex-selective property ofcomplex 1. Compared to conventional radiographic or luminescent assaysthat require multiple steps and/or the use of isotopically orfluorescently labeled nucleic acids, the present invention's label-freeapproach is more time and cost-effective as expensive and tediouspre-labeling or immobilization steps are avoided. On the other hand, thelabeling of an oligonucleotide with a fluorophore may disrupt theinteraction between the oligonucleotide with its cognate target.Finally, the present invention provides a label-free DNA-based detectionplatform employing luminescent transition metal complexes, which offerseveral advantages compared to the relatively more popular organicfluorophores, such as long phosphorescence lifetimes, large Stokes shiftvalues and modular syntheses. Additionally, the present invention isshown to effectively detect lead ions in tap water. The presentinvention's novel switch-on, label-free G-quadruplex-based luminescentdetection method for lead ions is a useful tool in environment and watersafety monitoring.

Detection Principle

The lead(II) ion is known to induce the structural transition of aguanine-rich single-stranded oligonucleotide into a G-quadruplex, whichis a non-canonical DNA secondary structure consisting of planar stacksof four guanines stabilized by Hoogsteen hydrogen bonding. In oneaspect, the present invention provides a label-free G-quadruplex-basedassay for the rapid detection of lead ions in aqueous solution usingiridium(III) complex 1. In a second aspect, the present inventionprovides a paper-based detection chip of the label-free G-quadruplexbased assay for portable and more convenient monitoring method (FIG. 8).In another embodiment of the present invention, the detection chip is afilter paper coated with patterned polydimethylsiloxane (PDMS) using atextured stamp, so the region of the paper coated with patterned PDMS issuperhydrophobic and the remaining area is hydrophilic. The hydrophilicarea consists of two parts, the detection zone and storage zone, andthey are linked by a hydrophilic channeL Then, a hydrophobic sticker ispasted between the two zones as a valve, the function of the hydrophobicsticker is to block the flow of the sample from the detection zone. Whenthe sticker is removed, the sample in detection zone will flow to thestorage zone (FIG. 11).

A number of paper-based platforms for lead ions have been reported. Forinstance, United States Patent US20130059391 A1, China PatentCN103487577 B and China Patent CN1687754 A, have reported that detectinglead ions by using testing strips. Besides, in the patent of WorldIntellectual Property Organization WO2012160857 A1 and WO2016090176 A1,the paper-based chips are coated with polymers. The present inventionincludes a removable hydrophobic sticker which acts as a valve to blockthe flow between the storage zone and detection zone (FIG. 11). Thepaper-based platform of the present invention enable the lead iondetection method to be portable and with detection time of no more thanten minutes. Moreover, the materials of hydrophobic sticker could beTeflon, propene polymer, polyethylene terephthalate and materials ofsimilar properties.

Experimental Section

Materials

Reagents, unless specified, are purchased from Sigma Aldrich (St. Louis,Mo.) and used as received. Iridium chloride hydrate (IrCl₃.xH₂O) ispurchased from Precious Metals Online (Australia). All oligonucleotidesare synthesized by Techdragon Inc. (Hong Kong, China).Polydimethylsiloxane (PDMS) prepolymer (RTV 615) is obtained fromMomentive Performance Materials (Waterford, N.Y.).

Fabrication of Paper-Based Detection Chip

FIG. 9(A) shows the process of fabricating the paper-based chip. ALiquid PDMS prepolymer (PDMS A) and a curing agent (PDMS B) are mixed in10:1 w/w ratio and spin-coated on a glass slide. A textured stamp isattached on the glass slide to obtain a PDMS coating on thepositive-relief features on the textured stamp. After that, the stamp isimprinted on a filter paper. The region on the paper touched by thetextured stamp becomes coated with PDMS to be a superhydrophobicpatterned coating; the remaining area is hydrophilic where themicrochannels are defined. The whole microchannel consists of two parts;one part is imprinted by patterned PDMS to become superhydrophobic. Theuntreated hydrophilic parts are used as detection zones as well asstorage zones. A hydrophobic polytetrafluoroethylene (Teflon) sticker ispasted between the two zones.

The single-stranded oligonucleotide (PS2.M, 100 μM, 2 μL) and Trisbuffer (200 mM Tris-HAc, pH 7.0, 10 μL) are added to the detection zone,while Complex I, (5 nM, 2 μL) is added to the storage zone.

Process of Detection (FIG. 11)

-   -   Four different concentrations of Pb²⁺ samples (not less than 1        μL) are added to the detection zones on two chips respectively.    -   The chip is heated at least 1 minute under temperature ranging        between 32° C. to 99° C., preferably 32.1° C. to 98.6° C. After        heating, the chip is then cooled down for at least 30 seconds        under temperature ranging between −18° C. to 32° C.    -   The sticker is removed and the fluorescence intensity (Emission        spectra are recorded in the 550-700 nm range using an excitation        wavelength range of between 200-400 nm.) is measured.

The results from the sample and the control (Pb²⁺, 50 nM) are compared.

Data Analysis

-   -   Images (FIG. 10 and FIG. 12) are analyzed by Image) 1.4        software.    -   The image type is changed to 8-bit gray scale.    -   A threshold of brightness is set to subtract the background.    -   The results of fluorescence intensity of the four water samples        are analyzed and displayed (FIG. 13).

A portable device is provided for the point of care testing of Pb²⁺, theresults can be semi-quantified by a smart phone or be quicklyqualitative analyzed by naked-eye observation. The portable device ismainly composed of a power, a heater, a UV light and a filter (FIG. 14).

INDUSTRIAL APPLICABILITY

The present invention relates to the synthesis and use of a luminescentIr(III) complex as a label-free G-quadruplex-based assay for thedetection of lead ions in a liquid or solution. In particular, thepresent invention describes a method for monitoring lead ions indrinking water.

If desired, the different functions discussed herein may be performed ina different order and/or concurrently with each other. Furthermore, ifdesired, one or more of the above-described functions may be optional ormay be combined.

Throughout this specification, unless the context requires otherwise,the word “comprise” or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers. It is also noted that in this disclosure and particularly inthe claims and/or paragraphs, terms such as “comprises”, “comprised”,“comprising” and the like can have the meaning attributed to it in U.S.Patent law; e.g., they can mean “includes”, “included”, “including”, andthe like; and that terms such as “consisting essentially of” and“consists essentially of” have the meaning ascribed to them in U.S.Patent law, e.g., they allow for elements not explicitly recited, butexclude elements that are found in the prior art or that affect a basicor novel characteristic of the invention. The term “chip” used in thespecification refers to but is not limited to a platform for detection,determination, evaluation and/or quantitative measurements of analytes.

Furthermore, throughout the specification and claims, unless the contextrequires otherwise, the word “include” or variations such as “includes”or “including”, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers.

Other definitions for selected terms used herein may be found within thedetailed description of the invention and apply throughout. Unlessotherwise defined, all other technical terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art towhich the invention belongs.

While the foregoing invention has been described with respect to variousembodiments and examples, it is understood that other embodiments arewithin the scope of the present invention as expressed in the followingclaims and their equivalents. Moreover, the above specific examples areto be construed as merely illustrative, and not limitative of thereminder of the disclosure in any way whatsoever. Without furtherelaboration, it is believed that one skilled in the art can, based onthe description herein, utilize the present invention to its fullestextent. All publications recited herein are hereby incorporated byreference in their entirety.

Citation or identification of any reference in this section or any othersection of this document shall not be construed as an admission thatsuch reference is available as prior art for the present application.

What we claim is:
 1. An apparatus for detecting lead ions in a liquidcomprising: at least one luminescent iridium(III) complex, wherein saidat least one luminescent iridium(III) complex is Ir(III) complex,[Ir(epyd)₂(delphen)]OF₆ wherein epyd=2-(4-ethylphenyl)pyridine; anddelphen=4,7-dichloro-1,10-phenanthroline; at least oneG-quadruplex-forming sequence; wherein said at least oneG-quadruplex-forming sequence is a single stranded oligomer in theabsence of lead ions and said at least one G-quadruplex-forming sequenceforms a G-quadruplex structure in the presence of lead ions and whereinsaid at least one G-quadruplex-forming sequence reacts with said atleast one luminescent iridium(III) complex to emit luminescent emission;and a detection chip comprises a plurality of superhydrophobic areas anda plurality of hydrophilic areas, wherein said hydrophilic areascomprise one or more detection zones where said at least oneG-quadruplex-forming sequence is stored, one of more storage zones wheresaid at least one luminescent iridium(III) complex is stored and one ormore connected zones that connect the one or more detection and one ormore storage zones, wherein said superhydrophobic areas comprise one ormore hydrophobic valves located between said one or more connected zoneswhich separate the one or more detection and one more storage zones; andwherein said one or more hydrophobic valves is removable to allow flowof said at least one luminescent iridium(III) complex from the at one ormore storage zones to the at one or more detection zones.
 2. Theapparatus according to claim 1 wherein said at least oneG-quadruplex-forming sequence is PS2.M, 5′-GTGGGTAGGGCGGGTTGG-3′ (SEQ IDNO. 1).
 3. The apparatus according to claim 1 wherein said liquid iswater.
 4. The apparatus according to claim 1 wherein said luminescentemission is measured by at least one spectrometer.
 5. A method fordetecting lead ions in a liquid comprising: providing the apparatus ofclaim 1; introducing said liquid to the one or more detection zone;removing the one or more hydrophobic valves; and measuring luminescentemission of the detection chip.
 6. The method of claim 5 furthercomprising incubating the detection chip after introducing the liquid tothe one or more detection zone at 32° C. to 99° C. and cooling thedetection chip at 18° C. to 32° C.
 7. The method according to claim 5wherein said at least one G-quadruplex-forming sequence is PS2.M,5′-GTGGGTAGGGCGGGTTGG-3′ (SEQ ID NO. 1).
 8. The method according toclaim 5 wherein said liquid is water.
 9. The method according to claim 5wherein luminescent emission is measured using at least one spectrometerwith an excitation wavelength of between 200-400 nm and the emissionspectrum is recorded in a wavelength of 550-700 nm.
 10. The methodaccording to claim 5, wherein said detection chip is a paper substrate.11. The method according to claim 10, wherein said superhydrophobicareas are coated by imprinting a solution of a PDMS prepolymer and acuring agent mixed in 10:1 w/w ratio.
 12. The method according to claim5, wherein the one or more hydrophobic valves comprisepolytetrafluoroethylene.